Transfusion-transmission of cytomegalovirus (TT-CMV) causes serious morbidity and mortality in immunocompromised recipients, including low birth weight infants (LBWI). Yeager first demonstrated in 1981 that TT-CMV could be mitigated in LBWI through transfusion of CMV-seronegative blood components, though the degree of mitigation could not be reproduced [1]. Substantial data (reviewed below) also shows that leukoreduction significantly mitigates TT-CMV with a residual risk approximating 2.4%, a figure similar to the residual risk of using CMV-seronegative components. There are no available data, however, to calculate the incidence of TT-CMV with the use of both CMV-seronegative + leukoreduced units for transfusion. Nonetheless, because of the serious risks CMV infection poses to this population, and policy decisions in many countries to implement (nearly) universal leukoreduction, the de facto clinical standard for transfusion support of LBWI has evolved to become CMV-seronegative + leukoreduced units. However, the "CMV-safety" of this approach has never been validated in a large prospective clinical trial. In fact, we continue to see cases of TTCMV in LBWI even when using these transfusion practices. Furthermore, it is not clear whether this represents the definitive/optimal transfusion approach for recipients at high risk for CMV transmission, or whether additional safety measures (CMV NAT to identify window-phase units;100% leukoreduction QC to identify units that failed leukoreduction) are needed to completely prevent TT-CMV. Thus, the investigators believe this to be a question of very high significance and put forth the hypothesis (explained in detail below) that the use of both CMV-seronegative + leukoreduced units will have a low residual incidence of TT-CMV (likely between 0.5 and 2.0%) and that the "cause" of TT-CMV can be explained by the presence of CMV DNA or excess leukocytes in the components. Project 2 represents a multicenter clinical cohort study, proposed in conjunction with additional basic laboratory studies, designed to investigate several important issues in prevention of TT-CMV. This study, and its cohort, have been designed and redesigned in a variety of iterations (including a randomized controlled trial) in consultation with NHLBI program staff, the Pediatric Committee of the Transfusion Medicine/Hemostasis Network of which the PI is a member of the Steering Committee, and nationally recognized biostatisticians. Additionally, this study design has been subjected to peer review at a national level. The rationale for the cohort trial design proposed in this application is described in detail in the text below. We are one of the few groups with access to a sufficiently large population of LBWI to accomplish such a study in a single geographic location, a point important in CMV transfusion studies due to latitudinal variations in CMV prevalence and serostatus. Also, the assembled investigators have: substantial blood center, hospital transfusion service and pediatric/neonatal transfusion expertise;significant experience with very sensitive highthroughput automated assays for quantitation of CMV DNA and residual leukocytes in filtered blood components (both developed and/or validated at Emory);and established relationships with highly trained and specialized neonatologists, all of which are deemed necessary to successful complete such a study. Thus, we believe that both the cohort approach and use of novel technologies are highly innovative approaches to address whether CMV-seronegative + leukoreduced blood components are optimally CMV-safe. Aim 1: To establish the effectiveness of combined CMV serology and leukoreduction to prevent TTCMV in at-risk LBWI born to CMV-seronegative and -seropositive mothers. In this prospective multicenter study, we will follow 1300 LBWI (650 each born to CMV-seronegative and -seropositive mothers), rigorously screened to eliminate cases of maternal CMV transmission, in order to calculate accurate determinations of the incidence of TT-CMV in high risk LBWI following uniform receipt of CMV-seronegative + leukoreduced blood. Aim 2: To detect CMV DNA and/or elevated residual WBC counts in blood components transfused to LBWI;to correlate positive results with episodes of breakthrough TT-CMV in the study population. Aliquots of all transfused components from Aim 1 will be screened by high throughput assays for CMV DNA (quantitative) and residual leukocytes. Episodes of documented TT-CMV in Aim 1 will be analyzed in the context of transfusions containing CMV DNA (including viral load) and/or elevated WBC counts (>5 x 106) to calculate the risk of TT-CMV with transfusion of units that are CMV DNA+ or failed leukoreduction, and whether breakthrough cases of TT-CMV could be prevented by CMV NAT and 100% leukoreduction QC. To date, we have: 1) developed and vetted the study protocol, 2) obtained consultation and preliminary agreement to proceed with IRB submission with IRB consultants suggesting approval will not be problematic, 3) entered into subcontract agreements in principle with the neonatologists and neonatology practices whose leadership is represented as either co-investigators in this application or as supporting key personnel, 4) added to our investigator group Dr. Ed Mocarski, who is now on faculty at Emory, a collaborator in our Center for Transfusion and Cellular Therapies (CTCT) laboratories, and recognized worldwide as a basic science and clinical leader and authority on CMV and its transmission both via transfusion and transplantation, 5) developed and validated novel methods for automated, accurate, high-throughput counting of residual WBCs in leukoreduced units in order to determine the incidence of filter failure, and whether inadequate filtration is associated with cases of TT-CMV, and 6) validated CMV NAT methods (in collaboration with Dr. Mocarski) for identifying potentially infectious blood components based on presence of CMV DMA, as well as infected patients following transfusion. Thus, we believe the tools are in place for successful completion of the proposed Specific Aims, which will represent the definitive analysis of the CMV safety of CMV-seronegative + leukoreduced transfusions, and will inform future transfusion decisions for these high risk populations. This work has not previously been done, and the results should lead to broader implementation of evidence-based transfusion strategies, improved blood inventory management, and decreased morbidity and mortality in the transfusion support of LBWI. Furthermore, this project addresses the PPG central theme since LBWI are very susceptible to opportunistic infections, like CMV, that are transmitted by transfusions and thus it represents a serious hazard of transfusion in this specialized patient population.